Determination of reduced glutathione in rat livers by HPLC-UVD

Objective To establish a high performance liquid chromatography with UV-absorbance detection method for determination of the level of reduced glutathione in rat livers.Methods After the liver tissue homogenate was made under the condition of ice bathing and tissue proteins precipitated by acetonitrile,the derivative reaction of the supernatant and fluorescent 4-fluoro-7-nitrobenzofuraza was carried out in borate buffer.The mobile phase was acetonitrile-0.02 mol/L potassium dihydrogen phosphate(23∶77,V/V),the flow rate was 1.0 mL/min,the detection wave-length was 470 nm and the injection volume was 10 μL.The reaction mixture was quantified with the external standard method.Results The linear range of reduced glutathione was 0.200-125 μg/mL(r=0.999 9,n=5),and the lowest limit of determination was 0.040 0 μg/mL(S/N=3).The recovery rate was 96.2%-104.5%,the intra-day and inter-day relative standard deviations were 1.1%-4.6% and 1.9%-7.5%,respectively.Conclusion This method is simple,rapid,accurate,sensitive and stable,which is suitable for determination of reduced glutathione in the liver.